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Review
. 2011 Jul 1;15(1):99-109.
doi: 10.1089/ars.2010.3564. Epub 2010 Dec 17.

Reduction of cysteine sulfinic acid in eukaryotic, typical 2-Cys peroxiredoxins by sulfiredoxin

W Todd Lowther  1 , Alexina C Haynes
Affiliations
Review

Reduction of cysteine sulfinic acid in eukaryotic, typical 2-Cys peroxiredoxins by sulfiredoxin

W Todd Lowther et al. Antioxid Redox Signal. .

Abstract

The eukaryotic, typical 2-Cys peroxiredoxins (Prxs) are inactivated by hyperoxidation of one of their active-site cysteine residues to cysteine sulfinic acid. This covalent modification is thought to enable hydrogen peroxide-mediated cell signaling and to act as a functional switch between a peroxidase and a high-molecular-weight chaperone. Moreover, hyperoxidation has been implicated in a variety of disease states associated with oxidative stress, including cancer and aging-associated pathologies. A repair enzyme, sulfiredoxin (Srx), reduces the sulfinic acid moiety by using an unusual ATP-dependent mechanism. In this process, the Prx molecule undergoes dramatic structural rearrangements to facilitate repair. Structural, kinetic, mutational, and mass spectrometry-based approaches have been used to dissect the molecular basis for Srx catalysis. The available data support the direct formation of Cys sulfinic acid phosphoryl ester and protein-based thiosulfinate intermediates. This review discusses the role of Srx in the reversal of Prx hyperoxidation, the questions raised concerning the reductant required for human Srx regeneration, and the deglutathionylating activity of Srx. The complex interplay between Prx hyperoxidation, other forms of Prx covalent modification, and the oligomeric state also are discussed.

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Figures

FIG. 1.
FIG. 1.
Typical 2-Cys peroxiredoxin catalytic cycle and hyperoxidation. Low levels of H2O2 are reduced by Prx through a pair of essential Cys residues, Cys-SPH and Cys-SRH. The sulfenic acid intermediate (Cys-SPOH) reacts with the Cys-SRH residue to form an intermolecular disulfide bond, which is subsequently reduced by thioredoxin. During this process, the Prx molecules alternate between dimeric and decameric states. The reduced, decameric form of the protein is the most reactive with H2O2 (51, 73, 75). As the level of H2O2 increases, eukaryotic Prxs can react with a second H2O2 molecule to form the sulfinic acid form (CysSPO2-) and, as a result, are inactivated. This hyperoxidation stabilizes the decameric state of the Prx molecule and can lead to the formation of filamentous and spherical, high-molecular-weight species; depicted schematically here. The molecular details of these interactions are unknown. Further oxidation of the Prx molecule to the Cys sulfonic acid form (CysSPO32-) can occur. Srx, however, can only reduce the CysSPO2- moiety.
FIG. 2.
FIG. 2.
Sequence alignment of representative sulfiredoxins. Murine, Drosophila, Arabidopsis, Nostoc species PCC7120 (a cyanobacterium), and S. cerevisiae Srxs show 91%, 60%, 43%, 41%, and 33% sequence identity to human Srx, respectively. The secondary structural elements for hSrx are shown above the alignment: α, α-helices; β, β-strands; η, 310 helices. The residues highlighted by the red background and white lettering are strictly conserved. Residues that are either conserved in the majority of the proteins or have conservative substitutions are boxed in blue and colored red. The black dots above the alignment indicate every tenth residue of human Srx. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).
FIG. 3.
FIG. 3.
Sulfiredoxin reaction mechanism and intermediates. The original mechanism, based on the analysis of S. cerevisiae Srx (gray shading), relies on the formation of sulfinic phosphoryl ester (Cys-SPO2PO32-) and a thiosulfinate intermediate (Prx-SPO-S-Srx) between the Srx and Prx molecules (6). Structural and biochemical data support the direct formation of the former intermediate (see text for details). The Srx-Prx thiosulfinate intermediate has been confirmed for the yeast and human enzyme systems (33, 55). On reduction of this thiosulfinate with GSH or Trx (R-SH), the repaired Prx molecule (Prx-SPOH) can return to the Prx catalytic cycle (long dashed lines). A recent study showed that yeast Srx, which contains an additional Cys residue within a loop insertion (Fig. 2; also see the regions highlighted in green in Fig. 4), can resolve the Srx-Prx thiosulfinate through the formation of an intramolecular disulfide bond [Srx-(S-S)] (56). Alternative reaction paths and intermediates between Srx, Prx, and GSH (short dashed lines and arrows) remain to be investigated.
FIG. 4.
FIG. 4.
Surface features and nucleotide-binding motif of sulfiredoxin. (A) Surface representation of the ATP•Mg2+ complex (PDB code 3CYI) (31). Residues lining the hydrophobic pockets near the γ-phosphate (orange) and Mg2+ ion (gray) are highlighted in white. Blue and red surface features indicate the nitrogen and oxygen atoms of the surface side chains. The location of the Cys-containing loop insertion in yeast Srx and Cys99 of human Srx are highlighted in green. (B) Closeup of the human Srx active site. The novel ATP-binding motif of Srx consists of Lys61, Ser64, Thr68, His100, and Arg101. Cys99 is located at the bottom of the active site ∼5 Å away from the γ-phosphate of ATP. In this image from an engineered Srx(C99A)•PrxI(C52D)•ATP•Mg2+ complex (PDB code 3HY2), Asp52 mimics the incoming sulfinic acid moiety (see text and Fig. 5 for additional details) (29). The Mg2+ ion and its associated water molecules are shown as gray and red spheres, respectively. The position of Cys99 (green) was modeled from the crystal structure of wild-type, human Srx in complex with ATP•Mg2+ in panel A. Pro73 and Asp74 have been labeled and colored green to indicate the location of the 17 residue, Cys-containing insert found in S. cerevisiae Srx (Fig. 2). (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).
FIG. 5.
FIG. 5.
The human Srx•PrxI complex. (A) Front and side views of the toroidal Srx-PrxI complex model containing 10 Prx (pink/purple) and 10 Srx molecules (blue/cyan) (28). (B) Surface representation of one Prx dimer and its active-site and backside interactions with two Srx molecules. (C) Close-up of the active-site interface in the Srx(C99A)•PrxI(C52D)•ATP•Mg2+ complex. Same coloring scheme used as in Fig. 4B. (D) Close-up of the backside interface highlighting the local secondary structure of the PrxI C-terminus. In this complex, the resolving Cys residue, Cys173, was mutated to Ser, indicated by the black dot in the sequence alignment. The white surface on the Srx molecule highlights conserved residues. Orange highlighting on PrxI indicates conserved residues that interact with Srx. The purple dots on the alignment denote those residues that are different for PrxIII. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).
FIG. 6.
FIG. 6.
Sites of covalent modification for human PrxI and PrxII. (A) The hyperoxidized PrxII decamer with each monomer represented in a different color (PDB code 1QMV) (58). (B) Close-up of one Prx dimer highlighting the monomer–monomer interface near the N-termini, labeled N. Sites of covalent modification in all panels are colored yellow. The Cys-SPH residue is present in the sulfinic acid form (Csd). Numbering scheme used: PrxI residue number/PrxII residue number. Ser32/31 and the N-termini are located on the back of the Prx dimer away from the Prx active sites. (C) Close-up of the active site. Tyr194/193, part of the YF motif, and Lys197/196 are proximal to the peroxidatic Cys residue. (D) The dimer–dimer interface. Thr90/89 can be phosphorylated. For reference, the mutation of Thr82/Cys83 to Glu (green) results in the disruption of the decamer into its dimeric constituents (28). (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).
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